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Cotton dehydrin similar gene and application thereof

A technology of dehydration and cotton, applied in application, genetic engineering, plant genetic improvement, etc., can solve problems such as production reduction

Inactive Publication Date: 2011-06-01
BIOCENTURY TRANSGENE CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Most crops are sensitive to adverse environmental conditions and often experience reduced yields due to environmental stress

Method used

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  • Cotton dehydrin similar gene and application thereof
  • Cotton dehydrin similar gene and application thereof
  • Cotton dehydrin similar gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1 Cloning of Cotton Dehydrin-like Gene GhDh1

[0033] 1. Processing of cotton material

[0034] The F1 generation seeds of Ezamian No. 11 germinated for 15 days, and the whole plant material was taken for 8 hours of drought induction, and frozen in a -70 degree refrigerator.

[0035] 2. Extraction of cotton total RNA

[0036] A. After grinding 0.1 g of cotton leaves with liquid nitrogen, add 0.5 ml of plant RNA extract (purchased from invitrogen), and shake until thoroughly mixed.

[0037] B. Leave at room temperature for 5 minutes.

[0038] C. Centrifuge at 12,000 rpm at 4°C for 1 minute, and transfer the supernatant to a new RNase-free centrifuge tube.

[0039] D. Add 0.1ml 5M NaCl and mix gently.

[0040] E. Add 0.3ml chloroform, mix up and down.

[0041] F. Centrifuge at 12,000 rpm at 4°C for 10 minutes, and transfer the upper aqueous phase to a new RNase-free centrifuge tube.

[0042] G. Add isopropanol equal to the volume of the obtained water, mix w...

Embodiment 2

[0152] Example 2 Construction of GhDh1 Gene Plant Expression Vector

[0153] see Figure 4 1. The GhDh1 gene cDNA amplified by PCR was ligated to pGEM-TEasy with T4 DNA ligase to obtain plasmid pGEM-GhDh1, pGEM-GhDh1 and pBI121 were simultaneously double-digested with BamH1 and SacI to obtain GhDh1 fragment and linear pBI121 vector respectively, and used T4 DNA ligase connects the GhDh1 fragment and the linear pBI121 vector to obtain the GhDh1 gene plant expression vector pBI121-GhDh1.

Embodiment 3

[0154] Example 3 Obtaining Transgenic GhDh1 Gene Tobacco Using Agrobacterium-mediated Transformation

[0155] 1. Preparation of Agrobacterium tumefaciens LBA4404 Competent Cells

[0156] 1) Pick a fresh LBA4404 single colony and inoculate it in LB liquid medium containing an appropriate amount of antibiotics, and culture it at 28°C until the logarithmic growth phase;

[0157] 2) Centrifuge at 8000rpm at 4°C for 5 minutes, and collect the bacteria in a small centrifuge tube;

[0158] 3) resuspend and wash the cells with 600 μl ice-cold 500 mM CaCl2;

[0159] 4) Centrifuge at 8000 rpm at 4°C for 5 minutes, add 100 μl of ice-cold 500 mM CaCl2 to the cell pellet, mix well and set aside (the effect is best after 24-48 hours).

[0160] 2. Transformation of Agrobacterium tumefaciens LBA4404

[0161] 1) Add 1 μl of plant expression vector plasmid DNA to Agrobacterium competent cells, mix gently, freeze in liquid nitrogen for 5 minutes, and warm at 37°C for 5 minutes;

[0162] 2) A...

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Abstract

The invention relates to the field of plant gene engineering, and provides a cotton dehydrin similar gene GhDh1. The nucleotide sequence of the cotton dehydrin similar gene GhDh1 is shown as SEQ ID No:1, a GhDh1 gene plant expression vector is constructed, tobacco is mediated and transformed by using agrobactrium tumefaciens to obtain GhDh1 transgenic tobacco, the GhDh1 transgenic tobacco is verified by adopting a drought simulation experiment, the result shows that the GhDh1 transgenic tobacco has stronger drought resistance compared with the contrast tobacco, and the expression of the GhDh1gene in the tobacco can improve the drought stress resistance of tobacco seeds in the germination process to a certain extent.

Description

technical field [0001] The invention relates to the field of plant genetic engineering, in particular to a cotton dehydrin-like gene, a plant expression vector constructed by the gene and its application in the development of drought-resistant, cold-resistant and salt-alkali-resistant transgenic plants. Background technique [0002] In nature, plants are exposed to a variety of environmental conditions, water deficit, cold and freezing, heat, salt and oxygen deficiency are the main environmental factors affecting plant growth. Most crops are sensitive to adverse environmental conditions and often experience reduced yields due to environmental stress. During the occurrence of stress, a series of physiological and biochemical changes will occur in plants, such as sugar, lipid, protein and nucleic acid, as well as photosynthesis, nitrogen fixation and respiration, etc. to adapt to the stress. Different stages of plant growth, including seed germination, seed maturation, and se...

Claims

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Application Information

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IPC IPC(8): C12N15/29C12N15/82C12N5/10C07K14/415A01H5/00
CPCC12N15/8273C07K14/415
Inventor 孙超陈文华王君丹崔洪志
Owner BIOCENTURY TRANSGENE CHINA
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